Project Summary/Abstract The thymus is the primary immune organ responsible for the generation of T cells, and its functional decline with age (involution) is considered a significant contributor to aging-associated immunosenescence. There is strong evidence that aging-related thymic involution is initiated during youth by mechanisms that operate in thymic epithelial cells (TECs) to result in their depletion and loss of compartmental organization. Despite its central role in the formation and maintenance of T cell adaptive immunity and the pleiotropic negative impacts of thymus involution during aging, surprisingly few molecular regulators of thymus organ function, homeostasis, and involution have been identified. A key transcription factor, FOXN1, is known to be a primary regulator of both thymus development and postnatal maintenance. However, the mechanisms of its regulation and the pathways through which it affects TEC development, function, and aging-related involution remain largely unknown. TECs are a small subset of total thymus cells and are notoriously difficult to isolate experimentally. Thus, identifying regulators and effectors by gene expression-based or biochemical analysis is difficult, and in any case would be specifically targeted to a TEC subset or specific age. Given the gradual nature of thymic involution, designing such experiments to identify key regulators of thymus function and maintenance with aging would be doubly challenging. Forward genetic screens are powerful tools for identifying genes based on their function, thus allowing unbiased discovery of novel pathways and mechanisms, but are not generally practical for aging-related phenotypes. In a recent R21-funded project, my lab performed a dominant modifier screen using ENU mutagenesis to identify novel mutations that, when heterozygous, either enhance (increase the severity) or suppress (rescue/reduce the severity) the Foxn1Z/Z thymic involution phenotype. We identified 18 dominant modifier lines, including both enhancers and suppressors. Because they impact Foxn1 expression and/or function in the Foxn1Z/Z model of premature thymic involution, our hypothesis is that these modifiers represent previously unrecognized genes that normally act to promote or restrain aging-related thymic involution. In the current proposal, we will follow up on our successful initial project to identify the modifiers found in our pilot screen, and test whether they affect the trajectory of involution during normal aging. This novel approach to investigating the molecular basis of thymus involution represents a potentially transformative approach to identifying new genes, pathways, and mechanisms that control thymus function and aging-related involution.